The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
The polymerase chain reaction (PCR) (Saiki et al., 1985) is by far the most important nucleic acid diagnostic tool. The first diagnostic tests based on PCR were quite cumbersome and not amenable to large scale screening methods (Isaksson et Landegren, 1999). Several post PCR steps, such as restriction enzyme analysis, agarose gel electrophoresis or heterogeneous hybridization assays were needed to confirm the identity of the PCR product (Kricka, 1999). Recently, the development of new fluorescent techniques has, however, led to novel assay formats that greatly simplify the protocols used for the detection of specific nucleic acid sequences. These methods involve the detection of a specific PCR product in a homogeneous solution without the need to open the amplification tubes after PCR (Williams et al., 1998). The results can be read in real time as the PCR product is accumulated or at the end of the thermal cycling protocol directly from the amplification wells. The choice between real time or end point measurement modes depends on whether a quantitative or qualitative assay is desired.
At the moment four technologies enabling specific sequence detection in a closed tube are commercially available. All of them (the TaqMan, Molecular Beacons, LightCycler and Amplifluor technologies) are based on fluorescence resonance energy transfer (FRET) (Clegg, 1992; Selvin, 1995). TaqMan (Holland et al. 1991; Lee et al., 1993) uses a linear probe that has a FRET donor in its 5′ end and an acceptor moiety at the 3′ end (Livak et al., 1995). When the probe is single stranded, the three dimensional conformation of the probe brings the two labels close enough to each other for the acceptor to quench the donor fluorescence. As the probe hybridizes to its target or when a DNA polymerase that has a 5′ to 3′ exonuclease activity cleaves it, the distance between the two labels increases enough for the donor fluorescence intensity to increase markedly. The difference between TaqMan probes and molecular beacons (Tyagi et Kramer, 1996) is that the optimized stem-loop structure of molecular beacons brings the two labels as close together as possible when the probe is not hybridized to a target sequence. This ensures maximal quenching efficiency. Upon hybridization to a target (or when cleaved by a DNA polymerase), the fluorescence intensity of the FRET donor increases as the physical contact between the two labels is disrupted. The Amplifluor technology (Nazarenko et al., 1997) utilizes hairpin shaped primers that basically function the same way molecular beacons do: when the primer is incorporated into a PCR product, the donor and quencher moieties are separated and donor fluorescence is thus increased. The Hybridization Probe format (Morrison, 1995) used in the LightCycler system (Wittwer et al, 1997a; Wittwer et al, 1997b) uses two adjacent probes that are labeled such that when both probes are hybridized to a target, the labels are brought close to each other and a FRET occurs between them. The sensitized acceptor emission is measured instead of the donor fluorescence.
All of these methods based on FRET are characterized by relatively high signal-to-noise ratios and a good ability to discriminate between positive and negative reactions. However, they are all limited in the sense that either a dual label probe or primer or two separate probes per each target have to be used. This seriously complicates probe design and synthesis. In addition, since they all employ labels with rapidly decaying fluorescence and broad emission peaks, the possibilities for multiplex detection are limited.
The two labels used in the homogenous assays described above are of different kind, i.e. one of the labels shall quench the other label as long as the probe to which they are attached is non-hybridized.